BioMed Central
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Virology Journal
Open Access
Review
Molecular advances in the cell biology of SARS-CoV and current
disease prevention strategies
Caren J Stark and CD Atreya*
Address: Division of Viral Products, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD 20892 USA
Email: Caren J Stark - ; CD Atreya* -
* Corresponding author
AntiviralsCell biologyMolecular virologySARS-CoVVaccines
Abstract
In the aftermath of the SARS epidemic, there has been significant progress in understanding the
molecular and cell biology of SARS-CoV. Some of the milestones are the availability of viral genome
sequence, identification of the viral receptor, development of an infectious cDNA clone, and the
identification of viral antigens that elicit neutralizing antibodies. However, there is still a large gap
in our understanding of how SARS-CoV interacts with the host cell and the rapidly changing viral
genome adds another variable to this equation. Now the SARS-CoV story has entered a new phase,
a search for preventive strategies and a cure for the disease. This review highlights the progress
made in identifying molecular aspects of SARS-CoV biology that is relevant in developing disease
prevention strategies. Authors conclude that development of successful SARS-CoV vaccines and
antivirals depends on the progress we make in these areas in the immediate future.
Introduction
Following reports of the last case of the severe acute respi-
ratory syndrome (SARS) epidemic in July 2003, there has
been remarkable progress in several areas of research on
the molecular identification of the pathogen and its
pathogenesis, replication, genetics, and host immuno-
genicity, as well as elegant epidemiological studies. The

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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coronaviruses such as HCoV-229E and HCoV-OC43 cause
only minor health problems such as the common cold
and gastrointestinal diseases. In contrast, the SARS-CoV
pathogen causes fever, pulmonary edema, and diffuse
alveolar damage in severely affected individuals (collec-
tively termed severe acute respiratory syndrome) [8].
SARS-CoV is also a unique coronavirus in that, to date, it
is the only member known to cause severe morbidity and
mortality in humans [8]. Demonstration that SARS-CoV
can cause serious public health problems has focused
attention on the need to understand the viral replicative
strategy and devise prophylactic measures.
The clinical symptoms of SARS are those of a lower respi-
ratory tract infection and are accompanied by damage to
the lungs [6,9,10]. Gastrointestinal involvement is also
common, with more than 20% of patients presenting with
watery diarrhea [11]. Fecal samples from SARS patients
taken up to 25 days after onset of disease contain viral
RNA, which suggests viral shedding through the bowels
[5]. Liver dysfunction has also been reported based on
observed necrosis in hepatocytes [9,12]. Post-mortem tis-
sue examination of SARS patients has found the virus
presence in lung, bowel, lymph node, liver, heart, kidney,
and skeletal muscle samples [13]. The primary mode of
SARS-CoV transmission is airborne via droplets [14,15].
However, there are also reports of the presence of replicat-
ing virus in blood cells (peripheral blood mononuclear

features of the disease [19-21]. Now the SARS-CoV story
has entered a new phase, a search for preventative strate-
gies and a cure. In this review, we highlight the progress
made in revealing the molecular aspects of SARS-CoV
biology and how such information may lead to strategies
for disease prevention.
Brief overview of the SARS-CoV genome
Coronaviruses are subdivided into three groups based on
genetic and serological markers [22]. Groups I, and II
infect mammals while group III is specific for avian spe-
cies. Group I members are the porcine transmissible gas-
troenteritis virus (TGEV) and epidemic diarrhea virus
(PEDV), feline and canine coronavirus (FCoV and CCoV),
and human coronavirus 229E (HCoV-229E). Group II
includes porcine hemagglutinating encephalomyelitis
virus (HEV), murine hepatitis virus (MHV), bovine,
equine, and rat coronavirus (BCoV, ECoV, and RtCoV),
and human coronavirus OC43 (HCoV-OC43). Group III
includes the turkey coronavirus (TCoV), pheasant corona-
virus and avian infectious bronchitis virus (IBV).
Although most closely related to Group II coronaviruses,
SARS-CoV, with some of its unique genetic features, repre-
sents a distinct phylogenetic group [22-24].
To date, approximately 61 SARS-CoV genomic sequences
have been analyzed representing different phases of the
epidemic (early, middle, and late) and two isolates
obtained from palm civets [18]. The SARS-CoV genomic
RNA is approximately 30 kb and is organized into 13 to
15 open reading frames (ORFs) [25-27]. The SARS CoV
structural gene arrangement follows the same pattern as

proceeds. Estimates put the error rate of an RdRp at 10
-3
to
10
-5
per nucleotide [30]. Coronaviruses also undergo high
rates of RNA recombination, providing an additional
mechanism by which the viruses can rapidly amplify
genomic diversity. The SARS-CoV polymerase gene has a
recombination breakpoint, suggesting multiple genetic
origins for this molecule. [31]. These evolutionary mech-
anisms may have facilitated the adaptation of the animal-
borne SARS-CoV ancestor to the human host, suggesting
that such events in the future could lead to a virus with
increased pathogenicity for humans or one capable of
infecting multiple species. Recent evidence indicates that
the human-adapted SARS virus has crossed into another
species. Sequence and epidemiological analyses revealed
that a SARS-CoV isolated from a pig was derived from a
human strain. Complete nucleotide sequencing of the pig
virus isolate (designated TJF) and an S gene-based phylo-
genetic tree analysis revealed a closer relationship with
human SARS-CoV isolates than with animal coronavi-
ruses [32].
Progress in cell biology of SARS-CoV: Signaling
pathways
Successful viral replication depends upon the ability of
the virus to subvert cellular processes to their advantage
and counteract cellular defense mechanisms. Such virus-
cell interactions represent potential targets for the devel-

pro-apoptotic [activation of p38 mitogen-activated pro-
tein kinase (MAPK)] and anti-apoptotic [activation of the
protein kinase B (PKB, also known as Akt)] signaling path-
ways, although Akt induction appears to be insufficient to
prevent the virus-induced apoptosis [37,38]. Exactly how
SARS-CoV manipulates these cellular signaling pathways
to facilitate viral replication remains to be determined.
As mentioned above, IL-8 induction was shown to be
dependent upon AP-1 activation by SARS-CoV S protein
and in this process NF-κB was not involved [34]. This may
partially explain the clinical observation of dramatic
cytokine storm (high serum levels of IL-6 and IL-8) and
inflammation responses observed in SARS patients in the
acute stage associated with lung lesions; it has been also
suggested that the elevations of IL-6 and IL-8 due to SARS-
CoV infection of the respiratory tract can induce the
hyper-innate inflammatory response [39]. It is established
that cellular MAPKs regulate AP-1 activation-dependent
IL-8 induction in viral infections [40-42]. In SARS-CoV
infection, the IL-8 induction signaling pathway is perhaps
related to angiotensin-converting enzyme 2 (ACE2), as
anti-ACE2 antibodies inhibit IL-8 induction/release [34].
ACE2 is the cellular receptor for the SARS-CoV and the
receptor-binding sites on the virion are located in the 12–
672 amino acid region of the S protein [43].
Current advances towards SARS-CoV
prevention strategies
During the SARS outbreak that occurred in 2002–2003,
the spread of the disease was primarily controlled by strict
quarantine protocols and patient-isolation measures as

ture presents an attractive target for tunnel binding antivi-
The balance of cell survival and cell death in response to SARS-CoV infectionFigure 1
The balance of cell survival and cell death in response to SARS-CoV infection. SARS-CoV is shown approaching a cell with
ACE2 receptors (blue "Y"s) on the surface. The virus enters the cell, uncoats, and the viral RNA is replicated and translated.
The SARS-CoV U122 protein induces apoptosis in cells. SARS-CoV S and N proteins each can activate the cellular AP-1 pro-
tein, which regulates apoptosis, as well as other cellular processes. AP-1 also activates IL-8, a cellular cytokine. SARS-CoV
infection induces both MAPK (pro-apoptotic) and Akt (anti-apoptotic) pathways. How this balance between cell survival and
apoptosis is maintained is yet unknown. Cellular proteins are labeled in blue, viral proteins in black.
Virology Journal 2005, 2:35 />Page 5 of 8
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ral drugs [55]. Finally, since the functional details of most
coronavirus replicase gene products are not known,
random screening of potential antiviral compound librar-
ies will be a key area of drug discovery for SARS virus in
the near future [47].
b. Vaccine development
Vaccines are the best and least expensive prophylactic
measures against pathogens that cause epidemics in
humans. The fact that high titers of virus neutralizing anti-
body to SARS-CoV are found in sera of patients recovering
from infection and that those infected with the virus show
improvement after passive antibody administration sug-
gests a SARS-CoV vaccine is possible and points toward
antibody based treatments for the disease [47,56-58].
However, in developing SARS CoV vaccines, there are les-
sons to be learned from the world of veterinary CoV vac-
cines. In a review by Saif, it was pointed out that
coronaviruses in general target mucosal surfaces and
therefore eliciting local (mucosal) immunity is a major
consideration in the development of SARS-CoV vaccines;

against the receptor-binding domain (RBD) present in the
S1 region of SARS-CoV. These antibodies effectively inhib-
ited the S-protein mediated SARS-pseudovirus entry up to
50%, suggesting the potential of the inactivated SARS-
CoV as antigen for vaccine development [61]. Depletion
of RBD-specific antibodies from patient or rabbit immune
sera by immunoadsorption, significantly reduced the
virus neutralizing ability of the sera, suggesting that the
RBD epitope in the S protein is a critical determinant in
developing vaccine strategies [62].
2.1. Cloned N protein
The N protein of SARS-CoV appears to be more conserved
than S and M proteins and it has been suggested that this
protein may play a role in cell-mediated immunity in
SARS-CoV infections and also is an important viral anti-
gen for the early diagnosis. Vaccination of C57BL/6 mice
with a SARS-CoV N protein expressed by an E1/partially
E3-deleted, replication-defective human adenovirus 5 vec-
tor was shown to produce potent SARS-CoV-specific
humoral and T cell-mediated immune responses, suggest-
ing the potential of this construct to be used as SARS-CoV
vaccine [63]. Along the same line, intra-muscular immu-
nization of BALB/c mice with a plasmid DNA construct
encoding the full-length N protein was shown to elicit
serum anti-N antibodies and spenocyte proliferative
responses against the N protein [64]. The immunized
mice also produced strong delayed-type hypersensitivity
(DTH) and CD8 (+) CTL responses to the N protein, sug-
gesting that the N protein is not only an important B cell
immunogen, but also can elicit broad-based cellular

3. Attenuated live virus
The third possibility is a genetically engineered version of
live SARS-CoV for traits such as attenuated phenotype,
increased immunogenicity, and safe handling (out of
BL3+ facility). A full-length SARS-CoV cDNA-containing
plasmid has been developed from which synthetic infec-
tious viral RNA can be produced [28]. This system allows
for the functional analysis of each gene in the context of
infection and can be used for making attenuated strains
for vaccine development.
Conclusions: Limitations to current SARS
vaccine strategies
SARS-CoV clearly has pandemic potential. Although
progress in SARS-CoV molecular and cell biology research
has been remarkable, there remain clear limitations
regarding vaccine development due to a lack of complete
understanding in the areas of animal models of the dis-
ease as well as host immune responses to the evolving
molecular diversity of this newly emerged human virus.
Caution is warranted when utilizing experimental data
originating from one SARS-CoV strain infection in one
animal species or cell line in the development of a human
vaccine. The rapid development of an effective SARS-CoV
vaccine depends upon continuing basic research.
A study on the evolving S protein molecular diversity in
SARS-CoV isolates and its unexpected profound immuno-
functional effects illustrates this point [68]. The S protein
exhibited minor genetic diversity among 8 strains trans-
mitted during human outbreaks in early 2003. Synthetic
versions of these S variants with human preferred codons

Acknowledgements
We thank Stephen Feinstone and Ron Lundquist of CBER, FDA for their
critiques and the National Vaccines Program Office (NVPO) for a grant to
CDA. CJS is supported by a postdoctoral fellowship administered by the
Oak Ridge Institute for Science and Education (ORISE).
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